Promyelocytic leukemia protein as a redox sensor

ABSTRACT

The present invention relates to a method for determining the redox status of a cell or tissue comprising a step consisting of determining the level of PML nuclear bodies in said cell or tissue.

FIELD OF THE INVENTION

The present invention relates to methods for determining the redoxstatus of a cell or tissue. More particularly, the present inventionrelates to promyelocytic leukemia protein as a redox sensor.

BACKGROUND OF THE INVENTION

Many diseases associated with human aging, including cancer,cardiovascular disorders, and neurodegenerative diseases have a strongoxidative stress component, but the basic molecular mechanisms thatconnect aging, age-related diseases, and oxidative stress remaininsufficiently understood.

Oxidative stress is the result of unregulated production of reactiveoxygen species (ROS), and cellular mismanagement of oxidation-reductionchemistry can trigger subsequent oxidative damage to tissue and organs.In particular, hydrogen peroxide is a major ROS by-product in livingorganisms and a common marker for oxidative stress. The chemical biologyof H2O2 is much more complex, however, as mounting evidence alsosupports a role for H2O2 as a second messenger in normal cellular signaltransduction. Peroxide bursts in response to cell receptor stimulationcan affect several classes of essential signaling proteins that controlcell proliferation and/or cell death. Included are kinases like themitogen-activated protein (MAP) kinase family, transcription factorssuch as nuclear factor [kappa]B (NF-[kappa]B), and activating protein 1(AP-1) as well as various protein tyrosine phosphatases (PTPs), ionchannels and G proteins. Despite the far-ranging consequences of H2O2 inhuman physiology and pathology, mechanistic details surroundingintracellular H2O2 generation, trafficking, and function remain elusiveeven in the simplest eukaryotic organisms.

Accordingly, interest in developing tools to study the physiological andpathological roles of H2O2 and related ROS in living systems iswidespread. For example, fluorescent probes are well suited to meet theneed for tools to map the spatial and temporal distribution of H2O2within cells. Such reagents have revolutionized the study of calcium inbiological systems and hold much promise for enhancing our understandingof H2O2 physiology and pathology. The major challenge for practical H2O2sensing in biological environments is creating water-soluble systemsthat respond to H2O2 selectively over competing cellular ROS such assuperoxide (O2-), nitric oxide (NO), and lipid peroxides. Several typesof small-molecule reporters have been described for H2O2 detection.Included are dihydro derivatives of common fluorescent dyes (e.g.,2′,7′-dichlorodihydrofluorescein, DCFH, and dihydrorhodamine 123, DHR),the Amplex Red/peroxidase system, phosphine-containing fluorophores,luminescent lanthanide complexes and chromophores with ROS-cleavableprotecting groups. Limitations of these and other currently availableresponsive probes include interfering background fluorescence fromcompeting ROS, potential side reactions with thiols that are present inhigh concentrations within cells, the need for external activatingenzyme, lack of membrane permeability, and/or lack of water solubilityor compatibility, requiring the use of organic co-solvents. Furthermore,these tools cannot be used on fixed or paraffin-embededtissues,precluding their use in most pathological human situations,prospectively or retrospectively.

Therefore there is a need for new reliable redox sensor for determiningthe redox status of a cell.

SUMMARY OF THE INVENTION

The present invention relates to a method for determining the redoxstatus of a cell or tissue comprising a step consisting of determiningthe level of promyelocytic leukemia protein (PML) nuclear bodies in saidcell or tissue.

A further aspect of the invention relates to a method for determiningthe redox status of a cell or tissue comprising a step consisting ofdetecting the formation of disulfide-linked PML complexes, PMLaggregation or formation of nuclear bodies in said cell or tissue.

DETAILED DESCRIPTION OF THE INVENTION

PML nuclear bodies (NBs) are matrix-associated domains that accumulatemany sumoylated proteins. How NBs are assembled, what is their functionand how As2O3 specifically triggers PML sumoylation remains unexplained.The inventors identify the redox potential as the critical determinantfor NB formation. Upon oxidant stress or As2O3 exposure, PML formsdisulfide-bond homodimers, which then multimerise in a matrix-associatedNB outer shell. Thus, PML represents a redox sensor, whose oxidationregulates NB-formation. This property explains the action or arsenic inAPL and pave the way for using PML to detect local changes in the redoxstatus in vivo.

Definitions

Throughout the specification, several terms are employed and are definedin the following paragraphs.

As used herein, the term “Reactive Oxygen Species” (ROS) generallyrefers to radicals and other non-radical reactive oxygen intermediatesthat can participate in reactions giving rise to free radicals or thatare damaging to organic substrates. Primary reactive oxygen species(ROS) such as superoxide radical, hydrogen peroxide, hydroxyl radicals,and ortho-quinone derivatives of catecholaranes exert their cellulareffects by modifying DNA, lipids, and proteins to form secondaryelectrophiles. Examples of such latter secondary electrophiles includehydroxyalkenals, nucleotide propenals, and hydroxyperoxy fatty acylchains. The secondary electrophiles are implicated in cellulardysfunction either because they are no longer able to participate innormal cellular activity or because they serve as electron acceptors inoxidative chain reactions that result in the modification of otheressential cellular components. Damage caused by the primary andsecondary ROS contributes to the etiology of human disease states causedby neuronal ischemia during stroke, post-cardiopulmonary bypasssyndrome, brain trauma, and status epilepticus.

As used herein, the term “oxidative stress,” generally refers to aphysiological state of a cell or tissue characterized by the generationof ROS that exceeds the ability of said cell or tissue to at leastpartially neutralize or eliminate them. The imbalance can result from alack of antioxidant capacity caused by disturbance in production,distribution, or by an overabundance of ROS.

As used herein, “redox state” and “redox status” are relative terms thatgenerally refer to the presence and relative concentration of freeradicals in a cell or tissue. Redox state influences oxidative stressexperienced by a cell or tissue and accordingly organ. Changes inoxidative stress can influence the redox status of the cells.

As used herein, the term ‘cell” refers to any eukaryotic cell.Eukaryotic cells include without limitation ovary cells, epithelialcells, circulating immune cells, cardiac cells, beta cells, hepatocytes,and neurons.

As used herein, the term “tissue”, when used in reference to a part of abody or of an organ, generally refers to an aggregation or collection ofmorphologically similar cells and associated accessory and support cellsand intercellular matter, including extracellular matrix material,vascular supply, and fluids, acting together to perform specificfunctions in the body. There are generally four basic types of tissue inanimals and humans including muscle, nerve, epithelial, and connectivetissues.

As used herein, the term “organ”, when used in reference to a part ofthe body of an animal or of a human generally refers to the collectionof cells, tissues, connective tissues, fluids and structures that arepart of a structure in an animal or a human that is capable ofperforming some specialized physiological function. Groups of organsconstitute one or more specialized body systems. The specializedfunction performed by an organ is typically essential to the life or tothe overall well-being of the animal or human. Non-limiting examples ofbody organs include the heart, lungs, kidney, ureter, urinary bladder,adrenal glands, pituitary gland, skin, prostate, uterus, reproductiveorgans (e.g., genitalia and accessory organs), liver, gall-bladder,brain, spinal cord, stomach, intestine, appendix, pancreas, lymph nodes,breast, salivary glands, lacrimal glands, eyes, spleen, thymus, bonemarrow. Non-limiting examples of body systems include the respiratory,circulatory, cardiovascular, lymphatic, immune, musculoskeletal,nervous, digestive, endocrine, exocrine, hepato-biliary, reproductive,and urinary systems. In animals, the organs are generally made up ofseveral tissues, one of which usually predominates, and determines theprincipal function of the organ.

As used herein the term “ Promyelocytic Leukemia protein” or “PML” isintended to include any molecule defined as such in the literature,comprising for example any types of PML and in particular, PML IV. ThePML gene consist of nine exons and several alternative spliced PMLtranscripts have been described (de Thé et al., 1991; Fagioli et al.,1992; Goddard et al., 1991; Kakizuka et al., 1991; Kastner et al.,1992). PML belongs to a family of proteins defined by the presence ofthe RBCC motif, a C3HC4 (RING finger) zinc binding motif, one or twoother cysteine-rich motifs, the B boxes and a coiled-coil region. PML islocalised in the nuclear diffuse fraction of the nucleoplasm and in adiscrete sub-nuclear compartment, the nuclear bodies (NBs) (Daniel etal., 1993; Dyck et al., 1994; Koken et al., 1994; Weis et al., 1994).The PML isoforms can be divided into seven groups, which we aredesignating as PML I-VII, on the basis of sequence differences, due toalternative splicing, at the C-terminus. It has been shown that PML IIIand PML IV exist without exon 5 (de Thé et al., 1991; Fagioli et al.,1992) and that PML V can exist without exons 5 and 6 or 4, 5 and 6(Fagioli et al., 1992), although the sequences for the latter twoisoforms are not available. Table I provides for the description of thePML isoforms with their corresponding accession numbers.

TABLE I Nomenclature for PML isoforms and alternative spliced variantsIsoforms References Accession PML I 882aa PML4 (Fagioli et al., 1992)882aa PML-1 (Goddard et al., 1991) M79462 882aa TRIM19 alpha (Reymond etal., 2001) AF230401 PML II 829aa PML2 (Fagioli et al., 1992) 824aa PML-3(Goddard et al., 1991) M79464 824aa TRIM19 gamma (Reymond et al., 2001)AF230403 854aa TRIM19 delta¹ (Reymond et al., 2001) AF230404 829aaTRIM19 kappa (Reymond et al., 2001) AF230410 PML III 641aa PML-L (de Theet al., 1991) SS0913 PML IIIa 593aa PML-S (de The et al., 1991) PML IV633aa PML3 (Fagioli et al., 1992) 633aa Myl (Kastner et al., 1992)X63131 633aa TRIM19 zeta (Reymond et al., 2001) AF230406 PML IVa 585aaTRIM19 lambda (Reymond et al., 2001) AF230411 PML V 611aa PML1 (Fagioliet al., 1992) 611aa PML-2 (Goddard et al., 1991) M79463 611aa TRIM19beta (Reymond et al., 2001) AF230402 PML VI 560aa PML-1 (Kakizuka etal., 1991) M73778 560aa PML-3B (Goddard et al., 1991) M80185 560aaTRIM19 epsilon (Reymond et al., 2001) AF230405 PML VIb 423aa TRIM19iota² (Reymond et al., 2001) AF230409 PML VIb 423aa TRIM19 eta² (Reymondet al., 2001) AF230407 PML VIIb 435aa TRIM19 theta (Reymond et al.,2001) AF230408

As used herein the term “PML nuclear bodies” or “NB” refers todonut-shaped nuclear domains containing PML protein (Hodges M, Tissot C,Howe K, Grimwade D, Freemont P S. Structure, organization, and dynamicsof promyelocytic leukemia protein nuclear bodies. Am J Hum Genet. 1998;63:297-304. doi: 10.1086/301991). PML nuclear bodies result from theaggregation of PML, notably by the formation of disulfide-linked PMLcomplexes.

The term “subject” as used herein denotes a mammal such as a rodent, afeline, a canine and a primate. Preferably, a subject according to theinvention is a human.

The term “healthy subjects” as used herein refers to a population ofsubjects who do not suffer from any known condition, and in particular,who are not affected with any disease that results from an oxidativestress.

The term “redox sensor”, as used herein, refers generally to a proteinthe expression of which in a cell or tissue can be detected by standardmethods in the art (as well as those disclosed herein), and ispredictive or denotes redox status in said cell or tissue.

Methods of the Invention

An aspect of the invention relates to a method for determining the redoxstatus of a cell or tissue comprising a step consisting of determiningthe level of PML nuclear bodies in said cell or tissue.

A further aspect of the invention relates to a method for determiningthe redox status of a cell or tissue comprising a step consisting ofdetecting the formation of disulfide-linked PML complexes, PMLaggregation or formation of nuclear bodies in said cell or tissue.

Determination of level of PML nuclear bodies or detection of theformation of disulfide-linked PML complexes, PML aggregation orformation of nuclear bodies may be performed by a variety of techniques.Generally, the methods involve contacting the cell with a bindingpartner capable of selectively interacting with PML. The binding partnermay be an antibody that may be polyclonal or monoclonal, preferablymonoclonal. In another embodiment, the binding partner may be anaptamer.

Polyclonal antibodies of the invention or a fragment thereof can beraised according to known methods by administering the appropriateantigen or epitope to a host animal selected, e.g., from pigs, cows,horses, rabbits, goats, sheep, and mice, among others. Various adjuvantsknown in the art can be used to enhance antibody production. Althoughantibodies useful in practicing the invention can be polyclonal,monoclonal antibodies are preferred.

Monoclonal antibodies of the invention or a fragment thereof can beprepared and isolated using any technique that provides for theproduction of antibody molecules by continuous cell lines in culture.Techniques for production and isolation include but are not limited tothe hybridoma technique, the human B-cell hybridoma technique and theEBV-hybridoma technique.

Alternatively, techniques described for the production of single chainantibodies (see e.g. U.S. Pat. No. 4,946,778) can be adapted to produceanti-PML, single chain antibodies. Antibodies useful in practicing thepresent invention also include anti-PML fragments including but notlimited to F(ab′)2 fragments, which can be generated by pepsin digestionof an intact antibody molecule, and Fab fragments, which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab and/or scFv expression libraries can be constructedto allow rapid identification of fragments having the desiredspecificity to PML. For example, phage display of antibodies may beused. In such a method, single-chain Fv (scFv) or Fab fragments areexpressed on the surface of a suitable bacteriophage, e. g., M13.Briefly, spleen cells of a suitable host, e. g., mouse, that has beenimmunized with a protein are removed. The coding regions of the VL andVH chains are obtained from those cells that are producing the desiredantibody against the protein. These coding regions are then fused to aterminus of a phage sequence. Once the phage is inserted into a suitablecarrier, e. g., bacteria, the phage displays the antibody fragment.Phage display of antibodies may also be provided by combinatorialmethods known to those skilled in the art. Antibody fragments displayedby a phage may then be used as part of an immunoassay.

Monoclonal antibodies for PML are described in the prior art. Forexample PGM3 is a commercially available antibody directed towards theN-terminus of the protein. Another example includes C7 antibody that iscommercially available from ABCAM.

In another embodiment, the binding partner may be an aptamer. Aptamersare a class of molecule that represents an alternative to antibodies interm of molecular recognition. Aptamers are oligonucleotide oroligopeptide sequences with the capacity to recognize virtually anyclass of target molecules with high affinity and specificity. Suchligands may be isolated through Systematic Evolution of Ligands byEXponential enrichment (SELEX) of a random sequence library, asdescribed in Tuerk C. 1997. The random sequence library is obtainable bycombinatorial chemical synthesis of DNA. In this library, each member isa linear oligomer, eventually chemically modified, of a unique sequence.Possible modifications, uses and advantages of this class of moleculeshave been reviewed in Jayasena S.D., 1999. Peptide aptamers consist ofconformationally constrained antibody variable regions displayed by aplatform protein, such as E. coli Thioredoxin A, that are selected fromcombinatorial libraries by two hybrid methods (Colas et al., 1996).

The binding partners of the invention such as antibodies or aptamers,may be labelled with a detectable molecule or substance, such as afluorescent molecule, a radioactive molecule or any others labels knownin the art. Labels are known in the art that generally provide (eitherdirectly or indirectly) a signal.

As used herein, the term “labelled”, with regard to the antibody, isintended to encompass direct labelling of the antibody or aptamer bycoupling (i.e., physically linking) a detectable substance, such as aradioactive agent or a fluorophore (e.g. fluorescein isothiocyanate(FITC) or phycoerythrin (PE) or Indocyanine (Cy5)) to the antibody oraptamer, as well as indirect labelling of the probe or antibody byreactivity with a detectable substance. An antibody or aptamer of theinvention may be labelled with a radioactive molecule by any methodknown in the art. For example radioactive molecules include but are notlimited radioactive atom for scintigraphic studies such as I123, I124,In111, Re186, Re188.

The aforementioned assays generally involve the binding of the bindingpartner (ie. Antibody or aptamer) to a solid support. Solid supportswhich can be used in the practice of the invention include substratessuch as nitrocellulose (e. g., in membrane or microtiter well form);polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex(e.g., beads or microtiter plates); polyvinylidine fluoride; diazotizedpaper; nylon membranes; activated beads, magnetically responsive beads,and the like.

The level of PML nuclear bodies (or detection of the formation ofdisulfide-linked PML complexes, PML aggregation or formation of nuclearbodies) may be determined by any well known techniques in the art.

For example an immunohistochemistry (IHC) method may be used. IHCspecifically provides a method of detecting targets in a sample ortissue specimen in situ. The overall cellular integrity of the sample ismaintained in IHC, thus allowing detection of both the presence andlocation of the targets of interest. Typically a sample is fixed withformalin, embedded in paraffin and cut into sections for staining andsubsequent inspection by light microscopy. Frozen samples may also beused. Current methods of IHC use either direct labeling or secondaryantibody-based or hapten-based labeling. Examples of known IHC systemsinclude, for example, EnVision™ (DakoCytomation), Powervision(R)(Immunovision, Springdale, Ariz.), the NBA™ kit (Zymed LaboratoriesInc., South San Francisco, Calif.), HistoFine(R) (Nichirei Corp, Tokyo,Japan). In particular embodiment, a tissue section may be mounted on aslide or other support after incubation with antibodies directed againstthe proteins encoded by the genes of interest. Then, microscopicinspections in the sample mounted on a suitable solid support may beperformed. For the production of photomicrographs, sections comprisingsamples may be mounted on a glass slide or other planar support, tohighlight by selective staining the presence of the proteins ofinterest. Therefore IHC samples may include, for instance: (a)preparations comprising cumulus cells (b) fixed and embedded said cellsand (c) detecting the proteins of interest in said cells samples. Insome embodiments, an IHC staining procedure may comprise steps such as:cutting and trimming tissue, fixation, dehydration, paraffininfiltration, cutting in thin sections, mounting onto glass slides,baking, deparaffination, rehydration, antigen retrieval, blocking steps,applying primary antibodies, washing, applying secondary antibodies(optionally coupled to a suitable detectable label), washing, counterstaining, and microscopic examination.

Another method includes immunofluorescence. Immunofluorescence is atechnique allowing the visualization of a specific protein in cells ortissue sections by binding a specific antibody chemically conjugatedwith a fluorescent dye such as fluorescein isothiocyanate (FITC). Thereare two major types of immunofluorescence staining methods: 1) directimmunofluorescence staining in which the primary antibody is labeledwith fluorescence dye, and 2) indirect immunofluorescence staining inwhich a secondary antibody labeled with fluorochrome is used torecognize a primary antibody. Immunofluorescence staining can beperformed on cells fixed on slides and tissue sections.Immunofluorescence stained samples are examined under a fluorescencemicroscope or confocal microscope. Typically, primary, or direct,immunofluorescence uses a single antibody that is chemically linked to afluorophore. The antibody recognises the target molecule and binds toit, and the fluorophore it carries can be detected via microscope. Thistechnique has several advantages over the secondary (or indirect)protocol below because of the direct conjugation of the antibody to thefluorophore. This reduces the number of steps in the staining procedure,is therefore faster, and can avoid some issues with antibodycross-reactivity or non-specificity, which can lead to increasedbackground signal.

In one embodiment, the method of the invention further may comprise astep of comparing the level of PML nuclear bodies (or detection of theformation of disulfide-linked PML complexes, PML aggregation orformation of nuclear bodies) in the cell or tissue with a predeterminedthreshold value. Said comparison is indicative of the redox status insaid cell or tissue. For example, the predetermined value may the levelof PML nuclear bodies determined in a cell or tissue which does notundergo an oxidative stress. Therefore, the predetermined value may thelevel of PML nuclear bodies in a cell or tissue isolated from a healthysubject. In this case a higher level of PML nuclear bodies in the cellor tissue compared to the predetermined value is indicative of that thecell or tissue undergoes an oxidative stress.

Biochemical methods may also be useful especially for detecting theformation of disulfide-linked PML complexes. Typically immunoblottingassay under non-reducing conditions as described in Example 1 may besuitable.

The method of the invention is particularly suitable for determining theredox status of a whole subject, by determining step of determining theredox status of at least one cell or tissue sample obtained from saidsubject. In a preferred embodiment, said method involves using severalcell or tissue samples obtained from the subject.

The sample can be a biological fluid such as whole blood, plasma, serum,nasal secretions, sputum, saliva, urine, sweat, transdermal exudates,cerebrospinal fluid, or the like. Alternatively, sources of such samplesinclude muscle, eye, skin, gonads, lymph nodes, heart, brain, lung,liver, kidney, spleen, thymus, pancreas, solid tumors, macrophages,mammary glands, mesothelium, and the like. Cell samples include withoutlimitation ovary cells, epithelial cells, circulating immune cells,.beta cells, hepatocytes, and neurons.

The method of the invention is also particularly suitable to determinewhether a subject has or is predisposed to having a diseasecharacterized by a strong oxidative stress component. Actually, if notregulated properly, the excess ROS can damage the lipids, protein or DNAof a cell, altering its normal function and leading ultimately to thedevelopment of certain disease states. The etiology of diseasesinvolving oxidative stress is in part related to the damage caused bythe primary and secondary ROS. ROS contribute to the pathogenesis ofimportant human diseases caused by neuronal ischemia during stroke,post-cardiopulmonary bypass syndrome, brain trauma, and statusepilepticus. ROS are also involved in cardiac damage induced duringischemic heart disease, renal damage induced by ischemia and toxins aswell as in more chronic diseases such as the destruction of neurons inParkinson's disease, Amyloidosis, Prion disorders, Alzheimer's disease,and other chronic neurodegenerative disorders. Autoimmune diseases suchas the destruction of the islets of Langerhans of the endocrine pancreasin Diabetes Mellitus are also encompassed.

Accordingly a further aspect of the invention relates to a method oftesting a subject thought to have or be predisposed to having a diseasecharacterized by a strong oxidative stress component comprising the stepof determining the redox status a cell or tissue sample obtained fromsaid subject.

The method of the invention is also particularly suitable fordetermining the impact of a compound on the redox status of a cell ortissue. Therefore according to one embodiment the invention relates to amethod for determining the impact of a compound on the redox status of acell or tissue comprising the steps consisting of:

i) determining the redox status of a cell or tissue by performing themethod as above described

ii) contacting said cell or tissue with said compound

iii) determining the redox status of said cell or tissue after step ii)by performing the method as above described

iv) and comparing the redox status determined at step iii) with theredox status determined a step i).

In a particular embodiment, the cell at step ii) may be geneticallytransformed with at least one nucleic acid molecule encoding for PML.The term “transformation” means the introduction of a “foreign” (i.e.extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, sothat the host cell will express the introduced gene or sequence toproduce a desired substance, typically a protein or enzyme coded by theintroduced gene or sequence. A host cell that receives and expressesintroduced DNA or RNA bas been “transformed”. The nucleic acid moleculemay also include a fusion partner so that the recombinant polypeptide isexpressed as a fusion polypeptide consisting of PML fused to said fusionpartner. The main advantages of fusion partners are that they assistdetection of said fusion polypeptide and also enhance protein expressionlevels and overall yield. Typically, the fusion partner is greenfluorescent protein (GFP). This fusion partner serves as a fluorescent“tag” which allows the fusion polypeptide of the invention to bedetected by fluorescence microscopy. The GFP tag is useful whenassessing subcellular localization of the fusion polypeptide of theinvention.

Typically, said transformation may be performed by using any vector wellknown in the art. Examples of suitable vectors include replicatingplasmids comprising an origin of replication, or integrative plasmids,such as for instance pUC, pcDNA, pBR, and the like. Other examples ofviral vector include adenoviral, retroviral, herpes virus and AAVvectors. Such recombinant viruses may be produced by techniques known inthe art, such as by transfecting packaging cells or by transienttransfection with helper plasmids or viruses. Typical examples of viruspackaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293cells, etc. Detailed protocols for producing such replication-defectiverecombinant viruses may be found for instance in WO 95/14785, WO96/22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat.No. 4,861,719, U.S. Pat. No. 5,278,056 and WO 94/19478.

According to one embodiment, the compound may be natural or not.Accordingly, the compound may be selected from the group consisting ofchemical entities (e;g. a small organic molecule) or biological entities(such as protein, nucleic acid . . . ).

Actually, said method may be particularly suitable for toxicologicalassays, to determine the toxicological effect of a material (e;g.plastic . . . ;) or a drug.

The method may be also particularly suitable for screening drugs usefulfor reducing oxidative stress.

Therefore according to one embodiment, the present invention relates toa method for screening a drug for reducing oxidative stress comprisingthe steps consisting of:

i) providing a candidate compound

ii) contacting a cell or tissue with said candidate compound

iii) determining the level of PML nuclear bodies in said cell or tissue

iv) comparing the level of PML nuclear bodies measured at step iii) witha predetermined value

v) and selecting the candidate compound which induce a lower level ofPML nuclear bodies than the predetermined value.

The candidate compound may be of various origin, nature and composition.It may be any organic or inorganic substance, such as a lipid, peptide,polypeptide, nucleic acid, small molecule, etc., in isolated or inmixture with other substances. The compounds may be all or part of acombinatorial library of products, for instance.

According to one embodiment, the cell or tissue at step ii) may beisolated from a subject having a disease characterized by a strongoxidative stress component. In another embodiment, the cell or tissuemay be contacted with an agent known for inducing stress before, afteror simultaneously with the candidate compound. For example, said agentmay be H2O2, Paraquat or As2O3.

Alternatively, the cell at step ii) may be genetically transformed withat least one nucleic acid molecule encoding for PML or a fusionpolypeptide as above described.

The invention also includes gene delivery systems comprising a nucleicacid molecule of the invention, which can be used in gene therapy invivo or ex vivo. This includes for instance viral transfer vectors suchas those derived from retrovirus, adenovirus, adeno associated virus,lentivirus, which are conventionally used in gene therapy. This alsoincludes gene delivery systems comprising a nucleic acid molecule of theinvention and a non-viral gene delivery vehicle. Examples of non viralgene delivery vehicles include liposomes and polymers such aspolyethylenimines, cyclodextrins, histidine/lysine (HK) polymers, etc.

The candidate compound may be also compared with a compound well knownin the art for reducing oxidative stress such as an antioxidant.

The candidate compounds that have been positively selected at the end ofstep v) may be then subjected to further selection steps in view offurther assaying its in vivo properties. For example, the candidatecompound may be administered in animal models for a diseasecharacterized by a strong oxidative stress component.

The method of the invention is also particularly suitable for monitoringthe redox status of a cell or tissue at different time intervals.

The present invention is also particularly suitable for monitoring atreatment of a subject with a drug for reducing oxidative stresscomprising determining the redox status of a cell or tissue sampleobtained from the subject by performing the method of the invention, andoptionally, comparing said redox status with a predetermined valuerepresenting a predetermined stage of oxidative stress, the redox statusof said cell or tissue with respect to the predetermined valueindicating the evolution of the oxidative stress in said patient, andtherefore the degree of efficacy of the treatment.

A further aspect of the invention relates to PML a redox sensor.

A further aspect of the invention relates to PML for use in thetreatment of a disease characterized by a strong oxidative stresscomponent.

Said diseases include but are not limited to cancer, diseases caused byneuronal ischemia during stroke, post-cardiopulmonary bypass syndrome,brain trauma, and status epilepticus, cardiac damage induced duringischemic heart disease, renal damage induced by ischemia and toxins aswell as in more chronic diseases such as the destruction of neurons inParkinson's disease, Amyloidosis, Prion disorders, Alzheimer's disease,and other chronic neurodegenerative disorders. Autoimmune diseases suchas the destruction of the islets of Langerhans of the endocrine pancreasin Diabetes Mellitus are also encompassed.

Indeed without wishing to be bound by any theory, the inventors believethat PML represents a tampon for reactive oxygen species and thus mayeventually contribute to the control of the redox status of the celltissue or organ.

According to the invention it should be understood that those of skillin the art may use any protein or gene sequence variant as long as ithas the properties of PML. “Function-conservative variants” are those inwhich a given amino acid residue in a protein or enzyme has been changedwithout altering the overall conformation and function of thepolypeptide, including, but not limited to, replacement of an amino acidwith one having similar properties (such as, for example, polarity,hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, andthe like). Amino acids other than those indicated as conserved maydiffer in a protein so that the percent protein or amino acid sequencesimilarity between any two proteins of similar function may vary and maybe, for example, from 70% to 99% as determined according to an alignmentscheme such as by the Cluster Method, wherein similarity is based on theMEGALIGN algorithm. A “function-conservative variant” also includes apolypeptide which has at least 60% amino acid identity as determined byBLAST or FASTA algorithms, preferably at least 75%, most preferably atleast 85%, and even more preferably at least 90% , and which has thesame or substantially similar properties or functions as the native orparent protein to which it is compared.

In specific embodiments, it is contemplated that PML polypeptides usedin the therapeutic methods of the present invention may be modified inorder to improve their therapeutic efficacy. Such modification oftherapeutic compounds may be used to decrease toxicity, increasecirculatory time, or modify biodistribution. For example, the toxicityof potentially important therapeutic compounds can be decreasedsignificantly by combination with a variety of drug carrier vehiclesthat modify biodistribution.

According to the invention, PML polypeptides may be produced byconventional automated peptide synthesis methods or by recombinantexpression. General principles for designing and making proteins arewell known to those of skill in the art.

Another aspect of the invention relates to a nucleic acid moleculeencoding for PML for use in the treatment of a disease characterized bya strong oxidative stress component.

Typically, said nucleic acid is a DNA or RNA molecule, which may beincluded in any suitable vector, such as a plasmid, cosmid, episome,artificial chromosome, phage or a viral vector as above described.

So, a further object of the invention relates to a vector comprising anucleic acid encoding for PML for use in the treatment of a diseasecharacterized by a strong oxidative stress component.

A further object of the invention relates to a host cell comprising anucleic acid encoding for PML (or a vector comprising a nucleic acidthereof) for use in the treatment of a disease characterized by a strongoxidative stress component.

Alternatively, it may be particularly suitable to use an inducer of PMLexpression for treating a disease characterized by a strong oxidativestress.

As use herein, the term “inducer of Promyelocytic Leukemia protein (PML)expression” denotes a compound, natural or not, which has the capabilityto up regulate or activate the expression of a gene encoding for PML andconsequently the expression of the corresponding protein.

Accordingly, a further aspect of the invention relates to an inducer ofPML expression for use in the treatment of a disease characterized by astrong oxidative component.

In a particular embodiment, the inducer of PML expression is selectedfrom the group consisting of interferons, including all types ofinterferons such as alpha, beta, omega and gamma intereferons. In apreferred embodiment the inducer of PML expression includesinterferon-alpha.

An “interferon” or “IFN”, as used herein, is intended to include anymolecule defined as such in the literature, comprising for example anytypes of IFNs (type I and type II) and in particular, IFN-alpha,IFN-beta, INF-omega and IFN-gamma. The term interferon, as used herein,is also intended to encompass salts, functional derivatives, variants,muteins, fused proteins, analogs and active fragments thereof. Thepolypeptide sequences for human interferon-alpha are deposited indatabase under accession numbers: AAA 52716, AAA 52724, and AAA 52713.The polypeptide sequences for human interferon-beta are deposited indatabase under accession numbers AAC41702, NP_(—)002167, AAH 96152, AAH96153, AAH 96150, AAH 96151, AAH 69314, and AAH 36040. The polypeptidesequences for human interferon-gamma are deposited in database underaccession numbers AAB 59534, AAM 28885, CAA 44325, AAK 95388, CAA 00226,AAP 20100, AAP 20098, AAK 53058, and NP-000610.

In a preferred embodiment the interferon is interferon-alpha.Interferon-alpha includes, but is not limited to, recombinantinterferon-α2a (such as ROFERON® interferon available fromHoffman-LaRoche, Nutley, N.J.), interferon-α2b (such as Intron-Ainterferon available from Schering Corp., Kenilworth, N.J., USA), aconsensus interferon, and a purified interferon-αproduct.

According to the invention, the compounds as above described (PML,nucleic acid vectors, inducer of PML expression) may be formulated aspharmaceutical compositions. The pharmaceutical composition according tothe invention may further comprise a pharmaceutically carrier orexcipient. The term “pharmaceutically acceptable” refers to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to a mammal, especially ahuman, as appropriate. A pharmaceutically acceptable carrier orexcipient refers to a non-toxic solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.The compounds may be combined with pharmaceutically acceptableexcipients, and optionally sustained-release matrices, such asbiodegradable polymers, to form therapeutic compositions. In thepharmaceutical compositions of the present invention for oral,sublingual, subcutaneous, intramuscular, intravenous, transdermal, localor rectal administration, the active principle, alone or in combinationwith another active principle, can be administered in a unitadministration form, as a mixture with conventional pharmaceuticalsupports, to animals and human beings. Suitable unit administrationforms comprise oral-route forms such as tablets, gel capsules, powders,granules and oral suspensions or solutions, sublingual and buccaladministration forms, aerosols, implants, subcutaneous, transdermal,topical, intraperitoneal, intramuscular, intravenous, subdermal,transdermal, intrathecal and intranasal administration forms and rectaladministration forms.

It will be understood that the total daily usage of the compounds andcompositions of the present invention will be decided by the attendingphysician within the scope of sound medical judgment. The specifictherapeutically effective dose level for any particular patient willdepend upon a variety of factors including the disorder being treatedand the severity of the disorder; activity of the specific compoundemployed; the specific composition employed, the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and rate of excretion of the specific compoundemployed; the duration of the treatment; drugs used in combination orcoincidental with the specific polypeptide employed; and like factorswell known in the medical arts. For example, it is well known within theskill of the art to start doses of the compound at levels lower thanthose required to achieve the desired therapeutic effect and togradually increase the dosage until the desired effect is achieved.

A further aspect of the invention relates to a method of testing apatient thought to be predisposed to a disease characterized by a strongoxidative component, which comprises the step of analyzing a sample fromsaid patient for detecting the presence of a mutation in the geneencoding for PML and/or its associated promoter.

Typically, the sample may a blood sample.

Typical techniques for detecting a mutation in the gene encoding for PMLmay include restriction fragment length polymorphism, hybridisationtechniques, DNA sequencing, exonuclease resistance, microsequencing,solid phase extension using ddNTPs, extension in solution using ddNTPs,oligonucleotide assays, methods for detecting single nucleotidepolymorphism such as dynamic allele-specific hybridisation, ligationchain reaction, mini-sequencing, DNA “chips”, allele-specificoligonucleotide hybridisation with single or dual-labelled probes mergedwith PCR or with molecular beacons, and others.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

EXAMPLE 1 Material & Methods

Constructs and SiRNAs

PMLΔSIM, PML 3K/RΔSIM mutants and GFP-SIM were constructed by deletionor insertion of the PML SIM coding sequence (amino acids 556 to 566)with the Quickchange II site-mutagenesis kit (Qiagen) into pSG5-PML,pSG5-PML 3K/R, or pEGFP-N1. Expression vectors for PML PML, 3K/R andC60A mutants were described previously¹⁵ and PMLC212A, PML B1(C140/143/148A) were also constructed by mutagenesis on pSG5-PML orMSCV-PML. PMLΔCC corresponds to the deletion of the coiled coil motif(amino acids 216 to 333). Unless otherwise indicated, PML refers to thePML-III isoform. PML and Sp100 were fused with MBP in the pMalC2 vector.SUMO-1, 2 and 3 cDNA, deleted from the 5′ diglycine coding sequence,were amplified by PCR and cloned in the BglII/HindIII restriction sitesof pEGFP-N1. Fusions of the Daxx or Hipk2 SIM (amino acids 730 to 741and 857 to 871 respectively) to GFP (pEGFP-C1-SIM_(Daxx or HipK2)) anddeletion from the pSG5-Daxx were similarly constructed. TDG sumoylationsite (amino acids 325 to 335)³⁶ was inserted in pEGFP-SIM_((PML)) bymutagenesis. PML-III was cloned in pECFP-C1 and SUMO-1 in pEYFP-C1;fusions were then sub-cloned in the MSCV vector. SiRNAs against SUMO-swere described previously²⁵.

Cell Lines, Transfections and Treatments

HeLa, MRC5, CHO, COS, SaOS and Pml−/− MEF cells were cultured in 10%Fetal calf serum DMEM medium (Gibco). Plasmid transfections wereperformed in CHO or COS with Effectene transfection reagent (Qiagen).Stable CHO cell lines, expressing the PML isoform, or its mutants, wereobtained by cotransfection of pSG5-PMLΔSIM, pSG5-3K/RΔSIM, pSG5-C212A orpSG5-B1 with the Dsp-Hygro vector; selected for 2 weeks in 800 mg/mlhygromycin-containing media. Clones were selected for similar PMLexpression levels. PML WT, PML C60A, PMLΔCC and PML 3K/R stableexpressing cell lines were described previously²⁵. MRC5 or SaOS weregrown on coverslides and treated with 400 U/ml IFNg for 48 h and IFNafor 24 h to induce PML expression. Primary mouse APL blasts wereobtained from serially transplanted APLs¹⁶ and then cultured in RPMImedium, 10% FCS supplemented with IL-3, IL-6 and SCF (Gibco). Infectionof immortalized pml−/− MEFs or primary progenitors was performed withretroviruses produced by CaCl₂ transfection of Plat-E cells by with theMSCV-PML/RARA retroviral vector¹⁶. Pml−/− or +/+ MEF cells weresimilarly transduced with MSCV-PML or PML mutants.

As₂O₃ (Fluka) treatments were performed at 1 mM or 10 mM for 1 h. H₂O₂from SIGMA-Aldrich was performed for 30 min at 0.1, 1 or 10 mM asindicated, Phenylarsine oxide (PAO) was purchased from SIGMA,solubilized at 50 mg/ml in DMSO and used at 0.1 mM, 1 mM or 10 mM for 1h, CdCl₂ from Flucka, was used at a final concentration of 10 mM. Cellswere treated with 10 mM Lactacystin (Calbiochem) overnight, and with 50mg/ml ready-made cycloheximide (SIGMA) as indicated. Pre-treatments with100 mM N-ethyl maleimide (NEM) (SIGMA) for 30 min were carried outbefore adding As₂O₃ or H₂O₂. TC-FlAsH (FlAs) was from Molecular Probes;cells growing on coverslides were treated with 1 mM for 1 h, washed inPBS before PFA fixation. Dithiarsolan-biotin conjugate (arsenic-biotin)was kindly given by Kenneth L. Kirk⁵³, solubilized in DMSO to a 5 mMstock solution, and used on cell culture at 10 mM for 1 h.

Protein Analysis

Cell lysates were resolved on 7%, 3 to 10% gradient or 12% SDS-PAGE gelsand transferred onto nitrocellulose membranes. Detection was performedwith the chemi-luminescent substrate SuperSignal WestPico (Pearce),using previously described home made mouse and chicken anti-hPMLantibodies^(15,25), the PML isoform-specific antibodies werecharacterized previously²⁹. For analysis under non-reducing conditions,b-Mercapto-Ethanol or DTT was omitted from the standard Laemmli buffer.In situ nuclear matrix was prepared as for immunofluorescence, butfinally resuspended in Laemmli buffer. Purifications of 6M Guanidium-HCldenatured (His× 6)-tagged proteins were done with Ni-NTA-agarose fromQiagen, as previously²⁵. The purification of the arsenic-bound proteinswere performed from As-biotin treated CHO-PML or transiently pSG5-PML wtor C212A transfected COS cells. Cells were lysed in 2% SDS-containingRIPA with Protease Inhibitor Cocktail (PIC) (Roche Applied Science),denatured at 95° C. for 5 min to release nuclear matrix-associated PML.Streptavidin-agarose (Molecular Probes) was added to the 0.3% SDSlysates for 30 min to 1 h, washed three times in RIPA and arsenic boundproteins were eluted in Laemmli buffer.

Microscopy and Antibodies

Immunofluorescence, electronic microscopy and antibodies were previouslydescribed¹⁵. In situ nuclear matrix preparations were prepared asdescribed¹. Confocal analyses were performed on a LSM510 Meta lasermicroscope (Carl Zeiss MicroImaging, Inc.) with a plan apochromat 63×N.A.1.4 oil immersion objective and increased resolution images wereobtained with deconvolution software (Autodeblur, Image Quant) usingblind iterative algorithms.

The mouse anti-GFP antibody was from Roche Applied Science; Goatpolyclonal anti-Lamin B (M20), rabbit polyclonal anti-RXRa (D20) andanti-Daxx (M-112) were from Santa Cruz Biotechnology. Rabbit polyclonalanti-Daxx from G. Grosveld was used for Western blot analysis.Anti-SUMO-1 (mouse monoclonal anti-GMP-1 antibody) and Rabbit polyclonalanti-SUMO-2/3 antibodies were from Zymed Laboratories. Rabbit polyclonalanti-RARA115 was a kind gift of Pierre Chambon. All the antibodies wererevealed by AlexaFluor 488 or 594-labeled secondary antibodies fromMolecular Probes. Fluorescence was quantified with ImageJ software.

Fluorescence Recovery After Photobleaching

FRAP experiments were carried out on a LSM510 Meta confocal microscope.For recovery of CFP-PML and YFP-SUMO-1 in MEFs, 4 images before and 60after bleach were taken, with 10 sec between acquisitions. Bleach pulseswere performed 6 times in ROI containing one NB, with maximum laserintensities. Pinhole was adjusted to 1 and optimal laser power wasadjusted to minimize bleaching during recovery. For As₂O₃ treated cells,FRAP was assessed 8 min, 20 min, 30 min and 1 h after drug addition. ForSUMOΔGG-GFP and GFP-SIM fusions, FRAP was performed at zoom 20, maximumspeed scan (0.05 sec between acquisitions), 10 images were acquiredbefore bleach, 3 iterations were used to bleach, and time of recoverywas between 15 and 30 sec.

Animal Experiments

Mouse experiments were repeated 3 times. Animal handling was doneaccording to the guidelines of institutional animal care committees,using protocols approved by “Comité Régional d'Ethique ExpérimentationAnimale (CREEA) n°4”. 25 mg/kg Paraquat (SIGMA) was dailyintraperitoneally injected in PML/RARA and PML/RARA-S77A leukemic mice¹⁷(to be fully described elsewhere). In vivo imaging was performed using aXenogen IVIS100 facility to ensure similar tumor mass pre-treatment.Mice were treated with 150 mg/kg for 1 h before bone marrow cytospins(from FVB/NICO) and 0.5 mm tumor cryosections (NUDE/SWISS)immuno-histochemistry; CHO-PML xenografts were performed as before⁴².

Statistics

The two-sided t-test was performed to validate the significance of theobserved differences, which were considered statistically significantwhen P<0.05

EXAMPLE 2 PML Nuclear Bodies Biogenesis Involves PML Oxidation

Intermolecular disulfide bonds were previously implicated instabilization of the nuclear matrix. Interestingly, upon exposure to DTT(a di-thiol reductant), PML NB labelling was lost from in situ matrixpreparations, while lamin B staining was unaffected, suggesting thatdisulfide bonds tether PML onto the nuclear matrix. In PML transfectedcells, Western-blot analysis under non-reducing conditions revealed highmolecular weight species that disappeared in reducing conditions,suggesting the existence of disulfide-linked PML complexes. Todemonstrate that these complexes are covalent PML dimers, we transientlyco-expressed (His)× 6-PML-V with a CFP-PML-III fusion and analysed thelysates by Western-blot in non-reducing conditions with isoform-specificantibodies. A specific complex of the expected antibody reactivity forPML-III/PML-V heterodimers was observed, formally demonstrating that PMLforms disulfide-bond dimers. We then compared total and nuclear matrixextracts from transiently transfected CHO cells under non-reducingconditions and observed that the matrix fraction was dramaticallyenriched in covalent PML dimers. Similar results were obtained withPMLΔSIM, PML3KR or PML3KRΔSIM, again demonstrating thatmatrix-association is independent of PML SUMO/SIM interactions, but isassociated with PML oxidation.

Disulfide bonds are oxidant-sensitive. We thus treated transiently PML-or PML3KR-transfected cells with the ROS-inducers As2O3 or H2O2 andobserved a dramatic increase in covalent PML homodimers. As expected,the thiol alkylator N-ethyl-maleimine (NEM) abrogated As2O3 orH2O2-enhanced homodimer formation, but also disrupted PML NBs inuntreated cells. As ROS status is profoundly altered in cell culture, weexamined NBs in vivo in basal conditions and after acute induction ofROS. While prominent NBs were observed in CHO-PML cells grown inculture, NBs were undetectable when these same cells were grown asxenografts in nude mice under physiological oxygen conditions. However,a dramatic increase in NBs was observed when mice were treated withParaquat, which induces ROS formation. Identical results were obtainedwith endogenous murine pml in normal bone marrow cells. Thus,oxidation-triggered disulfide bonds in PML regulate its partitioningbetween the nucleoplasm and nuclear matrix associated NBs.

These experiments identify ROS as the key regulator of nucleoplasmic/NBpartitioning and NB biogenesis. Conversely, in amino acid-starvedcultures, acquired NB-defects were only corrected by cysteine,suggesting that intracellular —SH content exerts some control overNB-morphogenesis40. Most cell-lines show abundant NBs, likely reflectingthe high oxygen tension in culture. Absence of NBs in most normal cellsand tissues in vivo and their prominence in cells exposed to ROS, suchas monocytes, granulocytes or undergoing apoptosis, inflammation orearly transformation, are fully consistent with ROS being criticalregulators of NB-aggregation in vivo. Thus, PML could be a ROS sensorand NBs, biomarkers of redox status in vivo.

EXAMPLE 3 Matrix-Associated PML is Oxidized

The matrix-associated PML fraction is hypersumoylated. Conversely,deletion of the coiled-coil of PML abrogates its association to thenuclear matrix, NB formation, and sumoylation. Thus, whereas As2O3induces PML targeting to the matrix and NB formation independently ofsumoylation, matrix association contributes to basal or As2O3-enhancedPML sumoylation. We therefore questioned how As2O3 could promotetransfer from soluble diffuse nuclear PML to insoluble,matrix-associated NBs. As2O3 controls protein phosphorylation and ROSproduction. We could not confirm in our experimental system thatAs2O3-induced PML phosphorylation by ERK½ controls PML sumoylation. Wethen examined whether As2O3-induced ROS could promote NB formationthrough PML disulfide formation, as intermolecular disulfide bonds wereimplicated in stabilizing the nuclear matrix. We observed that treatmentwith DTT (a dithiol reductant) of nuclear matrix prepared in situdisrupted endogenous PML NBs, without affecting lamin-B stainingSimilarly, a 1 hr treatment of CHO cells stably expressing the PML-IIIisoform (CHO-PML) with the thiol alkylator N-ethylmaleimide (NEM)disrupted basal PML NBs. Finally, most CHO-PML cells cultured for 10days in the presence of the ROS scavenger N-acetyl cysteine (NAC) lostbasal NB formation. NBs were restored by a 3 day wash-out. Thus,cysteine residues and ROS regulate NB biogenesis.

NBs are matrix-associated domains. When analyzed in the absence ofreducing agents (DTT or βME), the nuclear matrix fraction of transientlyPML-transfected CHO cells entirely consisted of high molecular weightPML species. DTT disrupts disulfide bridges and may also reduce SOH orSOS linkages. The observation that these high molecular weight PMLcomplexes reversed to monomeric species upon reduction, stronglysuggested that matrix-associated PML consists of intermolecularlydisulfide-bound PML multimers. Accordingly, pretreatment of CHO cellswith NAC prior to PML transfection led to a dramatic decrease in theabundance of the high molecular weight PML forms. To formallydemonstrate the existence of covalent PML multimers, we transientlyexpressed (His)× 6-PML-V with or without CFP-PML-III (PML isoforms ofdifferent sizes) in the presence of As2O3. Denatured whole-cell lysateswere analyzed under nonreducing conditions with isoform-specificantibodies. In cotransfected cells only, several, rather than one, highmolecular weight complexes were detected with the PML-V-specificantibody. Only the highest species reacted with anti-PML-III, implyingthat these were PML-III/PML-V multimers. PML-multimerization was alsodemonstrated by immunoprecipitating SDS-denatured lysates withanti-PML-III-specific sera, followed by guanidinium denaturation andHis-purification of the (His)× 6-PML-V-containing complexes. Undernonreducing conditions, only cells expressing both PML isoforms yieldedhigh molecular weight PML-III- and PML-V-reactive conjugates, whichshifted to the monomeric state after reduction. Finally, massspectrometric analysis of high molecular weight PML complexes purifiedunder denaturing conditions from As2O3-treated cells consisted primarilyof PML peptides (54%). Other detected proteins are most likelycontaminants, given that the most abundant one represented <6% of thetotal peptides. Collectively, these analyses strongly argue againstheterodimer formation with a distinct protein. Thus, although we cannotrule out the existence of intramolecular disulfide linkages, these dataindicate that intermolecular PML crosslinking by ROS-induced disulfidesis associated with its presence in the nuclear matrix and withNB-formation.

We next investigated whether oxidants enhance disulfide-bound PMLmultimerization. A short exposure to therapeutic levels of As203 (or toother oxidants, such as H2O2 or CdCl2, massively increased the amountsof covalent PML multimers in CHO cells transiently overexpressing PML.In stable CHO transfectants, oxidants similarly induced PMLmultimerization, whereas arsenical also promoted PML sumoylation.Formation of covalent multimers in transiently PML-transfected CHO cellswas abrogated by pretreatment with NEM or NAC. If PML covalentmultimerization by ROS is indeed the primary event initiating As2O3effects, other oxidants should mimic As2O3-enhanced NB biogenesis. Yet,strong oxidants (CdCl2, H2O2 . . . ) disrupt NBs in cultured cells,possibly because these agents fully oxidize some critical cysteines intocysteic acid, precluding disulfide formation and thus dissociating NBs.We thus examined whether paraquat, an acute ROS inducer, would regulateNB formation in vivo, a setting where ROS levels are likely morecontrolled. Although CHO-PML cells grown ex vivo displayed prominentNBs, those were barely detectable when these cells were grown asxenografts in nude mice. This could suggest that the microenvironment(notably oxygen tension) in vivo does not favor NB formation, whereas exvivo culture under hyperoxic conditions evokes NB aggregation.Critically, paraquat or As2O3 treatment of mice bearing CHO-PMLxenografts elicited a dramatic increase in NB formation (containing bothPML and its partner daxx). Western blot analysis under nonreducingconditions showed the occurrence of PML multimers in CHO-PML xenograftsderived from oxidant-treated mice. Enhanced NB formation by endogenousmurine PML was also observed in bone marrow cells on treatment withparaquat or As2O3 treatment. Altogether, these data demonstrate that, invivo, ROS inducers mimic As2O3 as to the regulation of PML oxidation, NBformation, and partner recruitment.

We first demonstrate that ROS regulate NB-biogenesis in vivo, explainingthe abundance of NBs in multiple stress conditions or in cells exposedto high oxygen concentrations, such as endothelial cells ([Koken et al.,1995] and [Lallemand-Breitenbach and de Thé, 2010]). NBs are nuclearmatrix domains and disulfides were previously implicated in nuclearmatrix formation ([Kaufmann et al., 1991] and [Stuurman et al., 1992b]).PML is the first example of a protein organizing a nuclear domain in aROS-dependent manner, suggesting that PML is a ROS sensor. Both ROS andPML have been implicated in multiple biological processes, notably DNAdamage response, senescence, and stem cell self-renewal, as well as inthe fine-tuning of some critical signaling pathways, including HIF1a orPTEN/AKT ([Song et al., 2008], [Trotman et al., 2006], [Bernardi andPandolfi, 2007], [Ito et al., 2008] and [Pearson et al., 2000]). PML NBformation could thus mediate some effects of basal ROS. In cellulosesynthase, cysteines arranged in a zinc finger become engaged intomultiple intermolecular disulfides upon ROS exposure (Kurek et al.,2002). This ROS-induced, oxidation-mediated, transition is responsiblefor the action of herbicides on cellulose synthase activity. PML, whichharbors three zinc fingers, forms several disulfide bridges, likely allrequired for full matrix association and PML sumoylation. This couldexplain why, despite formation of some disulfide bridges, the C212Amutant exhibits a defective sumoylation. Matrix association and/orsumoylation might also require an interchain zinc finger (Callaghan etal., 2005), itself possibly involving C212.

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

Bernardi and Pandolfi, 2007 R. Bernardi and P. P. Pandolfi, Structure,dynamics and functions of promyelocytic leukaemia nuclear bodies, Nat.Rev. Mol. Cell Biol. 8 (2007), pp. 1006-1016.

Callaghan et al., 2005 A. J. Callaghan, Y. Redko, L. M. Murphy, J. G.Grossmann, D. Yates, E. Garman, L. L. Ilag, C. V. Robinson, M. F.Symmons, K. J. McDowall and B. F. Luisi, “Zn-link”: A metal-sharinginterface that organizes the quaternary structure and catalytic site ofthe endoribonuclease, RNase E, Biochemistry 44 (2005), pp. 4667-4675.

Ito et al., 2008 K. Ito, R. Bernardi, A. Morotti, S. Matsuoka, G.Saglio, Y. Ikeda, J. Rosenblatt, D. E. Avigan, J. Teruya-Feldstein andP. P. Pandolfi, PML targeting eradicates quiescent leukaemia-initiatingcells, Nature 453 (2008), pp. 1072-1078.

Kaufmann et al., 1991 S. H. Kaufmann, G. Brunet, B. Talbot, D. Lamarr,C. Dumas, J. H. Shaper and G. Poirier, Association of poly(ADP-ribose)polymerase with the nuclear matrix: The role of intermolecular disulfidebond formation, RNA retention, and cell type, Exp. Cell Res. 192 (1991),pp. 524-535.

Koken et al., 1995 M. H. M. Koken, G. Linares-Cruz, F. Quignon, A.Viron, M. K. Chelbi-Alix, J. Sobczak-Thépot, L. Juhlin, L. Degos, F.Calvo and H. de Thé, The PML growth-suppressor has an altered expressionin human oncogenesis, Oncogene 10 (1995), pp. 1315-1324. View Record inScopus|Cited By in Scopus (190)

Kurek et al., 2002 I. Kurek, Y. Kawagoe, D. Jacob-Wilk, M. Doblin and D.Delmer, Dimerization of cotton fiber cellulose synthase catalyticsubunits occurs via oxidation of the zinc-binding domains, Proc. Natl.Acad. Sci. USA 99 (2002), pp. 11109-11114.

Lallemand-Breitenbach and de Thé, 2010 V. Lallemand-Breitenbach and H.de Thé, PML nuclear bodies, Cold Spring Harb. Perspect. Biol. 2 (2010),p. a000661.

Pearson et al., 2000 M. Pearson, R. Carbone, C. Sebastiani, M. Cioce, M.Fagioli, S. Saito, Y. Higashimoto, E. Appella, S. Minucci, P. P.Pandolfi and P. G. Pelicci, PML regulates p53 acetylation and prematuresenescence induced by oncogenic Ras, Nature 406 (2000), pp. 207-210.

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Trotman et al., 2006 L. C. Trotman, A. Alimonti, P. P. Scaglioni, J. A.Koutcher, C. Cordon-Cardo and P. P. Pandolfi, Identification of a tumoursuppressor network opposing nuclear Akt function, Nature 441 (2006), pp.523-527.

1. A method for determining the redox status of a cell or tissuecomprising a step of determining the level of promyelocytic leukemiaprotein (PML) nuclear bodies in said cell or tissue.
 2. A method fordetermining the redox status of a cell or tissue comprising a step ofdetecting the formation of disulfide-linked PML complexes, PMLaggregation or formation of nuclear bodies in said cell or tissue.
 3. Amethod for determining the redox status of a whole subject comprising astep of determining the redox status of at least one cell or tissuesample obtained from said subject by performing in said cell or tissuesample one or both of: determining the level of promyelocytic leukemiaprotein (PML) nuclear bodies in said at least one cell or tissue sample;or detecting the formation of disulfide-linked PML complexes, PMLaggregation or formation of nuclear bodies in said at least one cell ortissue sample.
 4. A method of testing a subject thought to have or bepredisposed to having a disease characterized by a strong oxidativestress component comprising the step of determining the redox status ofa cell or tissue sample obtained from said subject by performing themethod according to claim
 3. 5. The method according to claim 4 whereinsaid disease is selected from the group consisting of cancer, diseasescaused by neuronal ischemia during stroke, post-cardiopulmonary bypasssyndrome, brain trauma, status epilepticus, cardiac damage inducedduring ischemic heart disease, renal damage induced by ischemia andtoxins, and chronic neurodegenerative disorders.
 6. A method fordetermining the impact of a compound on the redox status of a cell ortissue comprising the steps of: i) determining the redox status of acell or tissue by one or both of: determining the level of promyelocyticleukemia protein (PML) nuclear bodies in said cell or tissue; ordetecting the formation of disulfide-linked PML complexes, PMLaggregation or formation of nuclear bodies in said cell or tissue ii)contacting said cell or tissue with said compound iii) determining theredox status of said cell or tissue after step ii) by one or both of:determining the level of promyelocytic leukemia protein (PML) nuclearbodies in said cell or tissue; or detecting the formation ofdisulfide-linked PML complexes, PML aggregation or formation of nuclearbodies in said cell or tissue; and iv) comparing the redox statusdetermined at step iii) with the redox status determined in step i). 7.A method for screening a drug for reducing oxidative stress comprisingthe steps of: i) providing a candidate compound ii) contacting a cell ortissue with said candidate compound iii) determining the level ofpromyelocytic leukemia protein (PML) nuclear bodies in said cell ortissue iv) comparing the level of promyelocytic leukemia protein (PML)nuclear bodies measured at step iii) with a predetermined value, and v)selecting the candidate compound which induces a lower level ofpromyelocytic leukemia protein (PML) nuclear bodies than thepredetermined value.
 8. A method for monitoring a treatment of a subjectwith a drug for reducing oxidative stress comprising determining theredox status of a cell or tissue sample obtained from the subject byperforming the method according to claim 1 and optionally, comparingsaid redox status with a predetermined value representing apredetermined stage of oxidative stress, the redox status of said cellor tissue with respect to the predetermined value indicating theevolution of the oxidative stress in said patient, and therefore thedegree of efficacy of the treatment. 9-13. (canceled)
 14. A method oftesting a patient thought to be predisposed to a disease characterizedby a strong oxidative component, which comprises the step of analyzing asample from said patient for detecting the presence of a mutation in thegene encoding for PML and/or its associated promoter.
 15. The method ofclaim 5, wherein said chronic neurodegenerative disorder is selectedfrom the group consisting of Parkinson's disease, amyloidosis, priondisorders, and Alzheimer's disease.
 16. A method of treating a diseasecharacterized by a strong oxidative stress component in a subject inneed thereof, comprising the step of administering to said subject atherapeutically effective amount of a promyelocytic leukemia protein(PML), a nucleic acid encoding a PML, or an inducer of PML expression.17. The method of claim 16, wherein said inducer of PML is aninterferon.